Process for the preparation of l-(+)-glutamine



United States Patent O This invention relates to a process for thepreparation of L-(+)-glutamine which makes it possible to obtain on anindustrial scale and within good yield pure and optically homogeneousL(+)-glutamine without forma- Fee It has now been found that the simpleaddition of carbon disulfide prior to amidation of 'y-esters of glutamicacid (Formula II) almost entirely prevents the formation ofpyrrolidonecarboxylic acid (Formula III). The diammonium salt of theN-dithiocarboxy-v-ester of glutamic acid (IV) is first produced, but isnot easily isolated in a pure state. verted into the ammonium salt ofglutamine-dithiocarbamate (V) by addition of ammonia. These reactionscan be eiiected in anhydrous or aqueous solution and a lower alkanol(e.g. methanol, ethanol, propanol, butanol, etc.) can be used withadvantage as solvent. The best results are obtained With a 'methanolicsolution of the L-glutamic acid-y-methyl ester.

VIII NHC S-SNH4 VII tion or splitting off of a racemate. The process ischaracterized in that a v-ester of L-glutarnic acid is treated withammonia in the presence of carbon disulfide. L-(+)-glutamine is freedfrom the reaction product by slightly acidifying.

L-(+)-glutamine (Formula VII) is an important, naturally occurring aminoacid. Unlike other amino acids, for example methionine, lysine andL-glutamic acid, its scientific or technical application has hithertobeen greatly limited because of its high price. The high price is adirect result of the currently employed processes for its preparation.These involve a number of complicated and uneconomic operations. Thereis thus a real need for a simpler, less expensive process forindustrial-scale synthesis of L-( -glutamine.

Attempts have already been made, but without success, to convert thereadily available y-esters of L-glutamic acid (Formula II) directly intoamides. Such methods, however, always yield a preponderance ofpyrrolidonecarboxylic acid (Formula 111) [A. F. Beecham, J. Am. Chem.Soc. 76, 4615 (1954)]. Introduction and subsequent splitting off ofgroups for protection of the amino group has hitherto been economicallyunacceptable, rendering the synthesis of L-(+)-glutamine too expensive.

N Ha

R =1ower alkyl Thestability of the dithiocarbamoyl group in stronglybasic solution is Wholly unexpected. Another remarkable and unexpectedfeature is that the diammonium salt (IV) is formed much quicker than thexanthogenate (VIII) and that the equilibrium does not shift in favor ofthe formation of the ammonium salts of the pyrrolidonecarboxylic acid(III) and the xanthogenic acid (VIII), as the reaction proceeds.

Upon completion of the amidation, the excess of ammonia is removed andthe reaction mixture is rendered slightly acid by addition of an acid,preferably acetic acid. The free acid of the dithiocarboxy-glutamine(V1) is first formed and then splits spontaneously to give carbondisulfide and L-(+)-glutamine (VII). L-(+)-glutamine, isolated in goodyield, shows the same optical activity and possesses exactly the sameproperties as naturally occurring glutamine. It can be used as acomponent for artificial culture media and as medicament.

That the synthesis can be so readily accomplished without racemizationis rather remarkable, for it is known, that L-glutamic acid and itsderivatives easily racemize; and this process has a particular advantagein that it does not require the resolution of a racemic mixture ofintermediates into the active components.

The aforedescribed process may be modified as fol- This ammonium salt isthen con- 1000 grams of L-glutamic acid-'y-methyl ester are suspended ina mixture of 500 milliliters of carbon disulfide and 5 liters ofmethanol at and a current of NH is passed into saturation with cooling.The ester dissolves in a few minute. The reaction vessel is closed andthe mixture is allowed to stand for 3 to days at 20; it is then filteredand the excess ammonia is removed under reduced pressure at atemperature not exceeding 35. After acidifying with 4 liters of 4 Naqueous acetic acid, the solution is warmed at 60 for 30 minutes and thecarbon disulfide which separates out is distilled 01?. The solution iscooled and enough methanol is added to raise the content of methanol to70 vol. percent. The solution is cooled to -5 for 2 hours and L-(+)-glutarnine which crystallizes out is filtered off and washed withmethanol and ether. For recrystallization the L-(+)-glutarnine israpidly dissolved in 3.5 liters of boiling water, 7 liters of methanolare added, and the solution'is cooled to 5 for 2 hours. In this way 650grams of pure L-(+)- glutamine, M.P. 187 (with decomposition), and [1x]32 (0.:3; HCl 1 N), are obtained.

Example 2 1000 grams of L-glutamic acid are dissolved in a solution of500 milliliters of 96 percent sulfuric acid and liters of methanol at 10and the solution is left for 4 hours at While cooling strongly, there ispassed in a current of ammonia at 0 until neutralization is effected.500 milliliters of carbon disulfide are then added and more ammonia ispassed in to saturation. After allowing to stand at 20 for 3 to 5 daysin a closed container, the excess of ammonia is removed under reducedpressure and filtration is effected. After acidification of the filtratewith aqueous acetic acid, the further procedure described in Example 1is followed. Yield and properties of the end product are the same asdescribed in Example 1.

Example 3 1000 grams of L-glutamic acid are dissolved in a solution of400 grams of anhydrous hydrogen chloride in 10 liters of methanol at 10and after 5 hours at 20 the so-obtained methanolic solution ofL-glutamic acid-vmethyl ester is worked up in the manner described inExample 2.

In the foregoing examples, other lower alkyl y-esters (e.g. the ethyl,propyl or butyl, etc. ester) may be used in lieu of the methyl ester.

What is claimed is:

1. A process for the preparation of L-(+)-glutamine comprising (a)reacting, with cooling to about 0- C., a lower alkyly-ester ofL-glutamic acid with ammonia and carbon disulfide, in a solvent selectedfrom the group consisting of water and a lower alkanol,

(b) maintaining the resultant solution for 3 to 5 days at about 20 C.,

(c) acidifying the resultant solution of amidified diammonium salt ofthe N-dithiocarboxy-v-ester of glutarnic acid of the formula 1sufficiently to free L-(+)-glutamine therefrom, and

(d) separating the free L-(+)-glutamine from the reaction mixture. 2. Aprocess according to claim 1, wherein the 'y-ester of the L-glutamicacid is I-glutamic acid y-methyl ester.

3. A process according to claim 1, wherein the y'ester of L-glutamicacid is produced in the reaction solution itself by esterification ofglutamic acid with a lower alkanol, and wherein the resulting 'y-esterof L-glutamic' acid is reacted with ammonia in the presence of carbondisulfide without prior isolation and purification.

4. A process according to claim 3, wherein the alkanol added is methanoland the *y-ester of L-glutamic acid produced in the reaction mixture inL-glutamic acid-7 the resulting ammonia-free mixture, distilling offcarbon disulfide, and crystallizing L-(+)--glutamine from th mixture.

References Cited in the file of this patent UNITED STATES PATENTS2,762,841 Vassel Sept. 11, 1956 2,768,966 7 Vassel Oct. 30, 19562,883,399

Amiard Apr. 21, 1959 OTHER REFERENCES Karrer: Organic Chemistry, page132 (1950).

1. A PROCESS FOR THE PREPARATION OF L-(+)-GLUTAMINE COMPRISING (A)REACTING, WITH COOLING TO ABOUT 0*C., A LOWER ALKYL-$-ESTER OFL-GLUTAMIC ACID WITH AMMONIA AND CARBON DISULFIDE, IN A SOLVENT SELECTEDFROM THE GROUP CONSISTING OF WATER AND A LOWER ALKANOL, (B) MAINTAININGTHE RESULTANT SOLUTION FOR 3 TO 5 DAYS AT ABOUT 20*C., (C) ACIDIFYINGTHE RESULTANT SOLUTION OF AMIDIFIED DIAMMONIUM SALT OF THEN-DITHIOCARBOXY-$-ESTER OF GLUTAMIC ACID OF THE FORMULA